Crosstalk between ECs and lung cancer cells causes chemoresistance in lung cancer spheroids
We began by investigating whether interactions between ECs and lung cancer cells cause chemoresistance in lung cancer spheroids. First, we collected conditioned media (CM) from three different cell cultures: human umbilical vein endothelial cells (HUVECs/ECs), non-small cell lung cancer (NSCLC) cells (NCI-H460 or A549), and lung cancer cells co-cultured with HUVECs. We then added the three different CM to lung cancer spheroids in 3D cell culture conditions. We next compared the sensitivities of spheroids cultured in each medium to two anticancer drugs (gefitinib and cisplatin) [Figure 1-A].
To detect cell death in the spheroids, we used the nucleic acid stain EthD-1, which emits fluorescence upon binding the DNA of dead cells. Treatment with the EGFR inhibitor gefitinib significantly reduced cell survival in lung cancer spheroids grown in CM from cultured HUVECs or lung cancer cells, whereas spheroids grown in CM from lung cancer cells co-cultured with HUVECs showed strong resistance to gefitinib [Figure 1-B].
We also evaluated the efficacy of cisplatin, a member of the platinum-based antineoplastic family of drugs, under the same conditions. Cisplatin also sufficiently increased the intensity of EthD-1 staining in lung cancer spheroids grown in CM from cultured HUVECs or lung cancer cells. However, cisplatin did not alter cancer cell viability in spheroids grown in CM from lung cancer cells co-cultured with HUVECs [Figure 1-C].
Taken together, these results suggest that factors secreted in response to crosstalk between ECs and lung cancer cells may play pivotal roles in chemoresistance in lung cancer spheroids.
HYOU1 expression is increased by factors secreted in response to crosstalk between ECs and lung cancer cells
We next sought to identify the factors inducing chemoresistance in lung cancer spheroids grown in CM from lung cancer cells co-cultured with HUVECs. To accomplish this, we subjected spheroids generated via the procedure shown in Fig. 1A to microarray analysis.
Analyses of the functional enrichment of genes with an absolute change of greater than 2-fold were performed using FunRich software. The microarray data revealed that 42 genes were significantly enriched and 103 genes were depleted in lung cancer spheroids grown in CM from lung cancer cells co-cultured with HUVECs compared to spheroids grown in CM from lung cancer cells or HUVECs alone [Figure 2-A, Supplementary Table I ].
Further, spheroids grown in CM from lung cancer cells co-cultured with HUVECs exhibited a greater than 1.5-fold increase in the expression of genes involved in gene expression, tRNA aminoacylation, and the unfolded protein response (UPR), whereas the expression of genes involved in the cell cycle and DNA replication were decreased [Figure 2-B]. In terms of biological processes, we observed the enriched expression of genes involved in the protein metabolism and energy pathways, whereas we observed decreased expression of genes involved in cell communication and nucleic acid metabolism [Figure 2-C].
Thirteen genes (AARS, CARS, CASC5, CENPE, CENPQ, CENPU, GFPT1, HYOU1, IARS, MARS, MCM10, SARS, SGOL2) were detected in all three analyses performed (DAVID, FUNRICH, and GSEA). Because gene expression related to tRNA aminoacylation and the UPR was significantly enriched in spheroids grown in CM from lung cancer cells co-cultured with HUVECs, we focused our efforts on aminoacyl-tRNA synthetase and HYOU1.
Western blot analysis showed that aminoacyl-tRNA synthetase protein expression in lung cancer spheroids was not changed by factors secreted in response to crosstalk between ECs and lung cancer cells. As the PI3K/AKT signaling pathway plays a major role in cell proliferation, cell survival, and invasion in cancer, we next investigated the effects of HYOU1 on PI3K/AKT pathway components. HYOU1 protein expression and AKT phosphorylation at Ser473 were increased in H460 spheroids grown in CM from lung cancer cells co-cultured with HUVECs relative to H460 spheroids grown under normal conditions [Figure 2-D]. This result prompted us to focus on functional roles of HYOU1 in TME of lung cancer.
Reciprocal crosstalk between NSCLC cells and HUVECs causes increased HYOU1 expression in MCTSs
We further sought to ascertain whether the direct interaction between ECs and lung cancer cells affects HYOU1 expression in MCTSs. To accomplish this, we co-cultured lung cancer spheroids with ECs and/or lung cancer cells (NCI-H460 or A549) in 3D cell culture. Spheroids co-cultured with both ECs and lung cancer cells showed enhanced compactness compared to spheroids cultured with lung cancer cells alone [Figure 3-A]. Using the technique described in [Figure 2D], we next estimated HYOU1 expression and PI3K/AKT signaling activation in these spheroids. We observed increased HYOU1 expression and AKT phosphorylation (Ser473) in spheroids co-cultured with ECs and lung cancer cells compared to spheroids cultured with lung cancer cells alone.
Because HYOU1 is a well-known component of the endoplasmic reticulum (ER) chaperone network, we also explored whether induction of HYOU1 is dependent on the canonical UPR pathway by inducing ER stress in spheroids co-cultured with ECs and lung cancer cells. Unexpectedly, we observed decreased expression of the three major ER stress sensor proteins [inositol-requiring enzyme-1 (IRE1), PKR-like ER kinase, and activating transcription factor-6 (ATF6)] in spheroids co-cultured with ECs and lung cancer cells [Figure 3-B]. These results indicate that the increased HYOU1 expression observed in spheroids co-cultured with ECs and lung cancer cells is unrelated to the ER stress response.
Next, we investigated which cells express more HYOU1 upon the co-culture of spheroids with ECs and lung cancer cells. To this end, we detected HYOU1 expression and localization via multilayer image acquisition using fluorescence microscopy in lung cancer spheroids (NCI-H460 or A549) grown with HUVECs. Interestingly, HYOU1 expression was only observed in lung cancer spheroids and absent in HUVECs [Figure 3-C].
Our previous study revealed that crosstalk between NSCLC cells and HUVECs induced strong chemoresistance in MCTSs . In the present study, we investigated the effects of HYOU1 on the chemoresistance induced by the interaction between lung cancer cells and HUVECs in MCTSs. We generated tumor spheroids with suppressed HYOU1 expression by co-culturing HYOU1 siRNA-treated (siHYOU1) lung cancer cells with HUVECs, and treating the resulting spheroids with or without gefitinib or cisplatin. After MCTSs were allowed to form for 72 h, we assessed cell viability by measuring the levels of the apoptosis marker cleaved caspase-3. We observed that the suppression of HYOU1 expression in lung cancer cells caused an increase in the expression of cleaved caspase-3 in MCTSs [Figure 3-D]. These results show that depletion of HYOU1 in lung cancer cells affects the drug sensitivity of the TME.
Depletion of HYOU1 inhibits tumor growth, and the stemness and expression of EMT-related proteins in lung cancer cells
Because HYOU1 is an ER-associated chaperone induced by hypoxia [34–36], we investigated its expression in lung cancer spheroids, which exhibit hypoxia. Spheroid (3D) cultures derived from various lung cancer cells exhibited increased expression of HYOU1 relative to monolayer (2D) cultures [Figure 4-A].
According to analysis of data from The Cancer Genome Atlas program, HYOU1 is an unfavorable prognostic marker in renal and thyroid cancer, but is not prognostic in lung cancer. HYOU1 has a low specificity for expression in the lung cancer TME. Nevertheless, we examined whether HYOU1 controls lung cell growth to investigate the potential effects of altered HYOU1 expression on lung growth. Clonogenic survival was diminished by depletion of HYOU1 in H460 cells (40%), A549 cells (64.2%), and H1299 cells (46.2%) [Figure 4-B].
To elucidate the functional roles of HYOU1, we next examined whether HYOU1 controls tumor growth and metastasis in a spheroid model similar to the lung cancer TME.
The siRNA-mediated depletion of HYOU1 significantly increased cell death in H460, A549, and H1299 spheroids [Figure 4-C]. Expression of two apoptosis markers, cleaved PARP and cleaved caspase-3, were measured in H460 and A549 spheroids following HYOU1 depletion. The p38 MAPK is related to hypoxia-induced apoptosis [37, 38] and ERK/MAPK is involved in resistance to apoptosis under hypoxic conditions [39, 40]. Inhibition of HYOU1 expression induced p38 activation, but attenuated ERK activation, in H460 and A549 spheroids. However, suppressing HYOU1 expression did not alter the activation of p53 or HIF1 [Figure 4-D].
To identify the possible effects of HYOU1 on cancer stem cell (CSC) populations of lung cancer cells, HYOU1-depleted lung cancer cells were cultured under spheroid-forming conditions, and the resulting spheroid number and size were analyzed [Figure 5-A]. Inhibition of HYOU1 significantly attenuated the spheroid-forming capacity of lung cancer cells. As CD133 expression plays a critical role in the maintenance of stem-like properties in lung cancer [41, 42], depletion of HYOU1 decreased the expression of CD133 in lung cancer cells [Figure 5-B]. These results show that HYOU1 may also be involved in the propagation of CSCs in lung cancer.
Because CSCs have been associated with tumor initiation, therapeutic resistance, and metastasis, we next sought to determine whether inhibition of HYOU1 expression sensitizes lung cancer cells to anticancer therapies and thus enhances their efficacy. After lung cancer spheroids (NCI-H460 or A549) with or without siHYOU1 were subjected to gefitinib treatment, the intensity of EthD-1 staining in spheroids was measured. Depletion of HYOU1 expression markedly enhanced lung cancer spheroid chemosensitivity to gefitinib [Figure 5-C]. To investigate the effects of HYOU1 on cell migration, we also performed wound healing assays following HYOU1 depletion in lung cancer cells and found that lung cancer cell migration was attenuated by HYOU1 depletion [Figure 5-D]. We next measured the expression of epithelial-to-mesenchymal transition (EMT)-related proteins in HYOU1-depleted lung cancer cells. Expression of N-cadherin, α-SMA, vimentin, and collagen I were all decreased in HYOU1-depleted lung cancer cells [Figure 5-E]. These results demonstrate that HYOU1 plays a pivotal role not only in inhibition of tumor growth and stemness but also in enhancing the anticancer efficacy of the lung cancer TME.
HYOU1 controls tumor growth via the alteration of interferon signaling in lung cancer cells
To reveal the molecular mechanism by which HYOU1 affects lung tumor growth, we performed gene expression profiling on the HYOU1-depletion system in H460 cells. Using a fold difference cutoff of greater than 2.5-fold, we identified 44 genes that were differentially expressed between HYOU1-depleted H460 cells and normal H460 cells [Supplementary Table II, III]. According to the Reactome Pathway Database, HYOU1 is functionally involved in the UPR, chromatin organization, and interferon (IFN) signaling [Figure 6-A]. It is noticeable that expression of IFN-type I (IFN- α, β) were more increased than IFN-type II (IFN-γ) by depletion of HYOU1 expression. IFN- α, β have been found to be effective in reducing the growth of various tumor . Western analysis also showed that expression of IFN-type I (IFN- α, β) were increased by inhibition of HYOU1 expression during process of cell death in lung cancer cells [Figure 6-B].
HYOU1 expression is downregulated by the inhibition of the PI3K/AKT/mTOR pathway Although HYOU1 is a larger protein than GRP78, its overall structure is highly homologous to that of GRP78. Because the inhibition of the PI3K/AKT/mTOR signaling pathway suppresses GRP78 expression [42, 43], we evaluated the effect of activating the PI3K/AKT/mTOR pathway on HYOU1 expression. Treatment with the mTOR inhibitors Torin2 and WYE-132, and the potent PI3K inhibitors GDC-0032 and PKI-402, significantly inhibited HYOU1 expression in H460 and H1299 cells [Figure 7-A].
Because mTOR inhibitors could decrease an HYOU1 expression in H460 and H1299 cells, we next examined whether mTOR controls HYOU1 expression by using siRNAs for mTOR and HYOU1 in H460 and H1299 cells. Western blot analysis revealed that siRNA against mTOR efficiently depleted HYOU1 expression, whereas the inhibition of HYOU1 did not affect mTOR expression [Figure 7-B] and H1299 cells. These resutls suggest that mTOR could regulate the expression of HYOU1 in lung cancers.